The rst-line drug Imatinib (IM) has achieved a curative effect in most chronic myeloid leukemia (CML) patients, but drug resistance remains a problem. More alternative therapeutic strategies need to explore. In recent years, targeting dysregulated DNA repair mechanisms provided promising options for CML treatment. We discovered the versatile MDC1 interacted with γ-H2AX and 53BP1 in the early stage of the DNA damage response of cells. MDC1 was over-expressed in CML cell lines and patients' BMMCs. By knocking down MDC1, non-homologous end-joining (NHEJ) pathways were mainly inhibited, leading to an intense accumulation of unrepaired intracellular DNA damage and an apparent cell apoptosis promotion. Notably, targeting MDC1 further enhanced drug sensitivity in IM-resistant CML cells. Our work demonstrated that MDC1 is a prospective target for CML treatment through regulating DNA damage repair mechanism, and also an alternative option to cope with the current IM resistance dilemma. This study extends the understanding of regulating dysfunctional DNA repair mechanisms for cancer treatment.
Background The treatment of chronic myeloid leukemia (CML) is facing the dilemma of tyrosine kinase inhibitors (TKIs) resistance and disease recurrence. The dysfunctional DNA damage repair mechanism plays an essential role not only in the initiation and progression of hematological malignancies but also links to the development of TKI resistance. Deciphering the abnormally regulated DNA damage repair and proteins involved brings new insights into the therapy of leukemias. As a G2/M phase checkpoint kinase and a DNA damage repair checkpoint kinase engaged in the DNA damage response (DDR), along with an oncogenic driver present in various cancers, the particular involvement of Wee1 in DNA damage is far from clear. Deciphering its function and targeting it via modulating DNA repair pathways is important for improving our understanding of cancer treatment. Methods Wee1 expression was assessed in cell lines using RT-qPCR and western blot, and Wee1 knockdown efficacy was validated using RT-qPCR, western blot, and immunofluorescence. Wee1 function was investigated by CCK-8, colony formation, and flow cytometry assay in vitro. Wee1 role in DNA repair and its interactions with other proteins were then studied using western blot, immunofluorescence, and double plasmid-repair studies. Finally, the CCK-8 and flow cytometry assay was utilized to investigate Wee1 and imatinib’s synergistic effect, and a CML mouse model was constructed to study Wee1’s role in carcinogenesis in vivo. Results Wee1 was reported to respond quickly to DDR in an ATM-γH2AX-MDC1-dependent way upon DNA double-strand breaks (DSBs) occurrence, and it regulated homologous recombination by stimulating the recruitment of critical proteins RAD51/BRCA1 upon DSB sites. Wee1 was also revealed to be abnormally upregulated in CML cells. Further suppression of Wee1 not only causes cell cycle arrest and inhibits the proliferation of cancer cells but also enhances CML cell sensitivity to Imatinib in vitro and in vivo, possibly through an excessive accumulation of overall DSBs. Conclusion Wee1 is extensively involved in the DRR signaling and DSB repair pathway. Inhibiting abnormally elevated Wee1 benefits CML therapy in both IM-resistant and IM-sensitive cells. Our data demonstrated that Wee1 participated in promoting cell proliferation and imatinib resistance in chronic myeloid leukemia via regulating DNA damage repair dependent on ATM-γH2AX-MDC1. In the fight against CML, Wee1’s dysregulation in the DNA damage repair mechanism of CML pathogenesis makes it a viable therapeutic target in clinical applications.
Background The first-line drug Imatinib (IM) has achieved a curative effect in most chronic myeloid leukemia (CML) patients, but drug resistance remains a problem. More alternative therapeutic strategies need to explore. In recent years, targeting dysregulated DNA repair mechanisms provided promising options for CML treatment. Methods The Co-IP assay was used to analyze the proteins that interact with Mediator of DNA Damage Checkpoint 1 (MDC1). The repair assays were based on the reporter plasmids to study the repair ability of cells. We detected DNA damage mainly through the comet assay and immunofluorescence assays. RT-qPCR and western blot were performed to measure the expression of MDC1 in bone marrow mononuclear cells (BMMCs) from CML patients and cell lines. Clone formation assays were used to test the proliferation ability of CML cells. Flow cytometry and western blot were used to analyze cell apoptosis. Results In this study, we discovered the versatile MDC1 interacted with γ-H2AX and 53BP1 in the early stage of the DNA damage response of cells. MDC1 was over-expressed in CML cell lines and patients’ BMMCs. By knocking down MDC1, non-homologous end-joining (NHEJ) pathways were mainly inhibited, leading to an intense accumulation of unrepaired intracellular DNA damage and an apparent cell apoptosis promotion. Notably, targeting MDC1 further enhanced drug sensitivity in IM-resistant CML cells. Conclusions Our work demonstrated that MDC1 is a prospective target for CML treatment through regulating DNA damage repair mechanism, and also an alternative option to cope with the current IM resistance dilemma. This study extends the understanding of regulating dysfunctional DNA repair mechanisms for cancer treatment.
Background The BCR-ABL fusion protein is the key factor that results in the occurrence of chronic myeloid leukemia (CML). Imatinib (IM) is a targeted inhibitor of BCR-ABL to achieve complete remission. However, remission failure occurs due to acquired resistance caused by secondary BCR-ABL mutations, underlining the need for novel BCR-ABL-targeting strategies. Circular RNAs (circRNAs) derived from tumor-related genes have been revealed as possible therapeutic targets for relevant cancers in recent investigations. In CML, the roles of this kind of circRNA are yet obscure. Methods Firstly, RT-qPCR was used for determining circCRKL expression level in cell lines and clinical samples, RNase R and Actinomycin D were employed to verify the stability of circCRKL. Then shRNAs were designed to specifically knockdown circCRKL. The function of circCRKL in vitro was investigated using CCK-8, colony formation assay, and flow cytometry, while a CML mouse model was constructed to explore the function in vivo. Finally, a dual-luciferase reporter assay, RNA pull-down, RNA immunoprecipitation, and rescue experiments were conducted to investigate the mechanism of circCRKL functioning. Results Here, we determined circCRKL, which derives from CML-relevant gene CRKL, is over-expressed in BCR-ABL+ cells. Then we noticed knocking down circCRKL using shRNA lentivirus dampens the proliferation of BCR-ABL+ cells both in vitro and in vivo, and augments susceptibility of resistant cells to IM. Intriguingly, we observed that circCRKL has a considerable impact on the expression level of BCR-ABL. Mechanistically, circCRKL could behave like a decoy for miR-877-5p to enhance the BCR-ABL level, allowing BCR-ABL+ cells to maintain viability. Conclusions Overall, the current study uncovers that circCRKL is specifically expressed and regulates BCR-ABL expression level via decoying miR-877-5p in BCR-ABL+ cells, highlighting that targeting circCRKL along with imatinib treatment could be utilized as a potential therapeutic strategy for CML patients.
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